Apparatus and process for rapidly characterizing and differentiating large organic cells

ABSTRACT

To characterize and differentiate large organic cells rapidly, individual particles are illuminated with monochromatic radiation of a wave length comparable to the size of the cell, producing a differential light scattering pattern about the illuminated cell. The pattern is sensed, preferably in the disclosed apparatus, by an array of detectors, and the sensed pattern employed as an identification and characterization of the cell. The pattern may be analyzed, or selected portions of the pattern employed, to differentiate cells embodying different features. The apparatus and process is especially useful for rapid identification and differentiation of leucocytes and other types of mammalian cells, the radiation for such analyses preferably being infrared radiation. A preferred structure for individually illuminating such cells with radiation and for sensing their differential light scattering pattern is disclosed.

PRIOR RELATED APPLICATION AND PATENTS

The present invention is directed to the use of differential lightscattering to rapidly characterize and differentiate large organiccells, particularly leucocytes and other types of mammalian cells. Tothis end, a process, an apparatus, and a preferred sensor structure isdisclosed, and various modifications thereof are suggested. Priorrelated patents and an application assigned to the assignee of thisapplication include:

U.S. Pat. No.: 3,624,835

Title: Microparticle Analyzer Employing a Spherical Detector Array

Inventor: Philip J. Wyatt

Date of Issue: Nov. 30, 1971

U.S. Pat. No.: 3,770,351

Title: Optical Analyzer for Microparticles

Inventor: Philip J. Wyatt

Date of Issue: Nov. 6, 1973

U.S. Pat. No.: 3,730,842

Title: Process for Determining Bacterial Drug Sensitivity

Inventor: Philip J. Wyatt, et al.

Date of Issue: May 1, 1973

U.S. Pat. No.: 3,754,830

Title: Scattering Cell Employing Electrostatic Means for Supporting aParticle

Inventor: D. T. Phillips, et al.

Date of Issue: Aug. 28, 1973

U.S. Pat. No.: 3,928,140

Title: Apparatus and Process for Testing Microparticle Response to itsEnvironment

Inventor: Philip J. Wyatt, et al.

Date of Issue: Dec. 23, 1975

Co-pending application Ser. No. 139,366, now abandoned

Title: Light Scattering Photometer Recorder Unit

Inventor: H. H. Brooks, et al.

Date of Filing: May 3, 1971

BACKGROUND

For many years, there has been a need for a way to identify anddifferentiate large organic particles rapidly, particularly leucocytesand other types of mammalian cells. To this end, numerous physicaltechniques have been proposed, and machines and processes have beendeveloped that employ these techniques. Such techniques include chemicalstaining methods, chromatographic analytical methods and physicalmethods such as automated microscopic examination systems. All of thesetechniques are quite limited in their capabilities, in the population ofcells that they can analyze, and in their analytical speed. Moreimportantly, many of the techniques have been shown to be ofquestionable accuracy.

The present invention is directed to an apparatus and process forrapidly identifying, characterizing and differentiating cells,particularly leucocytes and other mammalian cells. It employs ananalytical technique known as differential light scattering, a techniquewhich has been shown to be capable of rapid and accurate analysis ofmicroparticles.

The terms "identifying", "characterizing" and "differentiating" cellsare used in describing the usefulness of the invention. "Identifying"means to determine what the cell is, e.g. a polymorphonuclear leucocyte,while "characterizing" describes its physical features, such as size,shape, and dielectric structure, and "differentiating" separates ordistinguishes different types of cells, such as sickling red blood cellsfrom normal red blood cells, or normal lymphocytes from abnormallymphocytes with inclusions, or cancerous squamous cells from theirnormal counterparts, etc.

Various systems have been described in the published literature thatemploy differential light scattering techniques to analyze mammaliancell systems. Such publications include "A Flow System Multi-AngleLight-Scattering Instrument For Cell Characterization" by G. C. Salzmanand others, this article appearing in the Journal of Clinical Chemistry,Vol. 21, No. 9, pgs. 1297 to 1304 (1975), and "Cell Classification byLaser Light Scattering: Identification and Separation of UnstainedLeucocytes" by G. C. Salzman and others which appeared in ActaCytologica, Vol. 19, No. 4, pgs. 374 through 377 (1975). These articlesdescribe systems which employ a monochromatic light beam to illuminatewater suspended mammalian cells, the resulting differential lightscattering pattern being quite complex. For that reason, an empiricalapproach was employed to determine which discrete areas of the overalldifferential light scattering pattern could be employed to isolate aspecimen population of certain characteristics from populations of othercharacteristics. Obviously, such empirical approaches are quite limited,both in their ability to handle various cell populations and in theirability to produce meaningful results.

SUMMARY OF THE INVENTION

The present invention provides an apparatus and process for rapidlycharacterizing and differentiating cells, particularly leucocytes andother types of mammalian cells. It employs a beam of polarized,monochromatic illumination that is of a wavelength which isapproximately equal to the size of the cells of interest in the cellpopulation to be analyzed. Individual cells of that population areilluminated by the beam, the cells of interest producing differentiallight scattering (DLS) patterns that exhibit resonant scatteringcharacteristics having relatively broad maxima and minima, i.e., eachmaxima and minima will extend over an angular range usually on the orderof 10 to 30 degrees. These patterns are neither so simple in shape norso complex in detail that little or no useful information can be gleanedfrom them. Rather, because they are produced by a resonant scatteringsystem, they embody sufficient significant features to characterizeaccurately each individually illuminated scatterer, particularly suchphysical features as its size, shape, and dielectric structure,permitting the scatterer to be accurately and unambigously identifiedand differentiated.

The practical application of light scattering phenomena to theidentification, characterization, and differentiation of large organicparticles begins with a conventional liquid suspension of such cells.This suspension is aerosolized by the apparatus, and in the preferredprocess, droplets are produced, some of which may contain a cell. Thesedroplets are examined and those which contain a cell are separated fromthe other droplets, then the liquid surrounding each cell is evaporatedto result in a stream of free, airborne cells. Each of these separatedcells is illuminated in sequence with a beam of polarized, monochromaticradiation of a wavelength approximately equal to the average size of thecells being examined, such as a wavelength in the infrared region for asuspension of mammalian cells. The illumination scattered by each cellis detected at a sufficient number of angular locations about the cellto provide a differential light scattering pattern characteristic of thephysical properties of the cell.

These successive DLS patterns are recorded and analyzed to identify,characterize, and differentiate the cells. As an example of such ananalysis, the patterns produced by individual cells may be processed todetermine certain intensity ratios, such as the ratios of the first peakintensity to the intensities of subsequent peaks, and then cells withsimilar numbers of peaks in their differential light scattering patternsmay be sorted in a multi-dimensional analysis based upon these ratios togroup cells of similar features. By means of such an analysis of varioustypes of known cells, unknown cells may be identified with known celltypes. The detectors used to determine the differential light scatteringpaterns need not subtend uniform solid angles nor be normalized to auniform dark current; by employing the same system throughout theanalysis, such differences as these will not affect the results. Othervariations in the disclosed apparatus and process are set forth in thedetailed description and noted in the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

Although there are many possible types of instrumentation configurationsthat will suitably measure, record, and analyze DLS patterns from largeparticles, in accordance with the teachings herein set forth, there arecertain basic elements that are required for a practical system. Theseinclude a means to handle cells and introduce them one at a time into alaser beam. The laser itself is preferably a plane polarized carbondioxide infrared source operating at about 10.6 μm, however, othersources producing a suitable wavelength may be used. The laser shouldpreferably be coplanar with the line of sight of an array of 10 to 50individual elements. Ideally, the number of detector elements requiredin the array, N, is given in terms of the vacuum wavelength, λ_(o), thelargest present particle diameter, D, the refractive index, n_(o), ofthe medium in which the measurement is made, and the angular rangespanned by the array θ, by the simple relation N≃[2πDn_(o) /λ_(o)+4]θ/180°. For mammalian cells illuminated with infrared radiation of10.6 μ m, this number lies between about 10 and 50. Although the arrayelements lie preferably on an arc subtending 100° or more, a sufficientDLS pattern may be obtained from other element configurations, e.g.wherein the elements are not on an arc, are not equidistant from thescattering particle, the angular range subtended is even less than 100°,or the detectors are not equidistantly spaced.

Such a preferred structure and system is illustrated in the accompanyingdrawings, in which:

FIG. 1 is a schematic diagram of the preferred apparatus;

FIG. 2 is a view in vertical cross-section of the cell handling portionof the preferred apparatus;

FIG. 3 is a view in horizontal cross-section of the detector housingshowing the detector array;

FIGS. 4 a-d present examples of some differential light scatteringpatterns in the infrared from particles with high water content;

FIG. 5 is a schematic diagram of the analytical system for the preferredapparatus;

FIG. 6 a-c are sets of views similar to FIG. 3 of other versions of thedetector array; and

FIG. 7 is a view in vertical cross-section of another detector housing.

DETAILED DESCRIPTION OF THE INVENTION

Introduction

Central to an understanding of this invention is an understanding ofdifferential light scattering. While the basic concept of differentiallight scattering is explained in many patents and publications,especially those by the present inventor, briefly it employs a polarizedmonochromatic radiation source to illuminate one or more particles, theparticles scattering the illumination in a way characteristic of theirphysical features, features such as size, shape and dielectricstructure. This pattern of scattered illumination may be sensed byrotating a collimated detector about the scatterer or using an array offixed detectors; the measured intensity may be recorded as a function ofdetector angle to plot a differential light scattering pattern.

Such differential light scattering patterns may contain a great deal ofinformation about the scatterer, or they may yield little or noinformation about the scatterer. For example, if the size of theparticle is quite small in relation to the wavelength of theilluminating beam, little or no variation will be exhibited in theintensity of the scattered radiation as a function of angle about theparticle. On the other hand, if the size of the particle is quite largein relation to the wavelength of the illuminating beam, a great manymaxima and minima will be exhibited in the scattering pattern.Interpreting such patterns to extract salient particle characteristicsis a major task.

This task may be simplified significantly by adjusting the wavelength ofthe illuminating monochromatic light source to be approximately equal tothe overall size of the illuminated particle. When such approximateequality exists, i.e., the particles are in the "resonance" region theresultant differential light scattering pattern will exhibit maxima andminima, yet it will not be so complex that many features directlycorrelated to particle structure are hopelessly lost in the detail.

The use of resonance scattering techniques is quite significant. Thedifferential light scattering patterns are complex functions of theparticle size, shape, orientation, and structure, as well as thepolarization and wavelength of the incident radiation. The most criticalscattering parameter is the normalized size; i.e.

    ρ=(πDn.sub.o /λ.sub.o)=ka                    (1)

where "a" is the mean particle radius, D(=2a) the correspondingdiameter, λ_(o) the vacuum wavelength of the incident radiation, andn_(o) the refractive index of the medium surrounding the particle.Variation of the size parameter, ρ, most affects the correspondingdifferential light scattering pattern, but size itself is certainly oneof the least important, and most ambiguous, parameters fordistinguishing anomalous cells from normal cells, or one type ofleucocyte from another, or one type of pollen from another. In addition,the size distributions invariably overlap. As ρ becomes very large, thedifferential light scattering patterns are overwhelmed by the additionalpeaks which are of little analytical importance.

If the DLS pattern is to be recorded by an array of N detector elements,it can be shown that the optimal number of such detectors spanning thecomplete angular range from 0° to 180° is given simply by

    N≃2ρ+4                                   (2)

In other words, from the intensity data recorded at the N suitablyspaced locations spanning the entire 180° range, the DLS patterns may bevery accurately interpolated between all these locations. If the angularrange of interest be less than this 180°, then the number of detectorelements may also be suitably reduced approximately by the ratio of therange spanned to 180°. Now for any particular measurement using such anarray to sense the DLS pattern, the largest number of detector elementsrequired is dictated entirely by the size of the largest particle ofinterest to be measured. Thus N should always be chosen to be about2ρ_(max) +4, where ρ_(max) corresponds to the normalized size of thelargest particle of interest expected in the suspension to be studied.For an airborne squamous cell of mean diameter 60 μm illuminated by aninfrared wavelength of 10.6 μm, the optimum number of detector elementswoulld be about

    (2π60/10.6)+4≃40                          (3)

At large scattering angles, internal structure will play a major role indetermining the variations of differential light scattering features.Indeed, this long-known fact, pointed out by the present inventor in1968 in Vol. 7 of Applied Optics pgs. 1879 to 1896, has been the basisfor the extensive differential light scattering programs being conductedat the Los Alamos Scientific Laboratory. By use of larger angledifferential light scattering measurements, the LASL group has hoped tobe able to differentiate among similar types of mammalian cells. Theirmeasurements have been made using He-Ne lasers operating at a visiblewavelength of 632.8 nm. Such wavelengths are extremely small compared tothe mean dimensions of the squamous cells examined. Although somesuccesses have been reported, the ultimate limit of such measurementsusing so short a wavelength may well have been reached already. Thereason for this is straightforward. At larger scattering angles, thedifferential light scattering patterns for such large values of ρ areintimately dependent on ρ through the parameter

    ξ=nρp                                               (4)

where n is the mean refractive index of the scattering particle dividedby n_(o). Since different types of mammalian cells would be expected tohave slightly different n values, though most importantly they fall intodifferent size domains, some separation based on nρ measurements wouldbe expected. The reported LASL differentiations using "clusteralgorithms" are fortuitous, but will be hard-pressed to distinguishbetween cells with comparable nρ values. Although they were apparentlyaware of the importance of using longer wavelength radiation, theproblems associated with infrared radiation could well have beendiscouraging.

The present invention is based upon the realization that differentiationand characterization of mammalian cells and other large organicparticles by differential light scattering techniques will be practicalif, and only if, the measurements are performed using infraredradiation, for at these resonance wavelengths the size of the particlesbeing illuminated is comparable to the wavelength of the illuminatingradiation. This may be confirmed by considering first the other two sizeregimes, the regimes in which particles are very small compared to theilluminating radiation and in which the particles are very largecompared to the illuminating radiation.

In the former case using, say, microwave radiation, it can be shown thatthere will be no angular variation in the scattered intensity.Accordingly, the intensity measurement need only be made at a singleangle, any angle. The absolute scattered intensity from a single "small"particle, if such could be practically measured, cannot be used toclassify or separate particles, since such a quantity is ambiguous;i.e., no single intensity value may be associated with a particularclass of mammalian cell. Slightly different normalized sizes, ρ, areeasily compensated by variations in mean relative refractive index, nand vice versa. Furthermore, any structural or shape differences betweendifferent classes of cells will have no single-valued effect upon thescattered intensity and thus these differences also will beundetectable.

In the latter case, using, say, visible light, when the size of theparticle is very large compared to the wavelength of the illuminatingradiation, the differential light scattering pattern will embody morescattering detail than is needed to account for the particle's importantstructural features. An example of such patterns and the immense detailthey present is illustrated by the patterns incorporated in the article"Scattering by Individual Transparent Spheres" by H. H. Blau and otherswhich appeared in Applied Optics, Vol. 9, pg. 2522 et seq. (1970). Theplethora of data for each measurement presented in that article hasresulted from an exceedingly simple scattering system: a sphericaldroplet of a uniform dialectric structure illuminated by polarizedmonochromatic radiation of a wavelength approximately one onehundredththe size of the particle.

The present invention is directed to the rapid identification,differentiation, and characterization of cells that are quite largecompared to the wavelength of visible light, particularly various typesof mammalian cells. These cells often incorporate a membrane of onedielectric structure surrounding an interior of another dielectricstructure which may include various particles and structural anomaliesof still other dielectric structures. The shape and size of thesevarious structures often are quite non-spherical, some for example beingplatelet shaped. Should visible illumination, i.e. radiation whosewavelength is very small compared to the mean diameter of theseparticles, be employed to obtain differential light scattering patternsfor such particles, not only would an enormous number of maxima andminima be present in such patterns, but also, even minute changes in anyof these structural characteristics will result in enormous changes inthe scattering patterns. These considerations illustrate the nearfutility of attempting to use such complex differential light scatteringpatterns to distinguish between particles of different composition. Yetmany have proposed and employ just such analyses.

As has been noted, this invention is directed to an apparatus andprocess for rapidly identifying and differentiating large organic cells,especially mammalian cells. Such cells typically range from a fewmicrometers in diameter to no more than a few tens of micrometers indiameter. These cells include leucocytes, erythrocyctes and squamouscells. If such individual cells are illuminated with a beam ofpolarized, monochromatic radiation of a wavelength on the order of, say,10 micrometers, then broad maxima and minima will be exhibited in thedifferential light scattering pattern produced by this system. Thisrelationship, one in which the size of the particles are in theso-called resonance region, produces patterns having features which arefar more easily correlated to structural differences of the scatterer.Modest changes in the particles' average features will result insignificant changes in the pattern in certain angular ranges, yet forparticles of comparable size, the overall patterns will be quitecomparable in shape. Moreover, while such patterns will includesufficient detail to permit accurate characterization anddifferentiation of various particles, they do not include so great anamount of detail as to mask or impede the accurate mathematicalinterpretation of the physical features of the scatterer. Thisrealization, that the illuminated particles and the wavelength of theilluminated beam should be of roughly comparable size, makes practicalthe rapid and unambiguous characterization and differentiation ofmammalian cells and other large organic cells. Also, it gives rise to arelatively simple, yet eminently practical, apparatus and process forperforming such analyses.

The necessity of this relationship dictates use of an infrared laser forthe analysis of mammalian cell systems. Mammalian cells incorporate, andusually are surrounded by, water-like fluids. This presents asignificant difficulty. The absorption coefficients of water in theinfrared region are very large. Thus, the water normally present in andaround such cells will play a major role in any resonance differentiallight scattering measurement from mammalian cells. When watersuspensions of cells are illuminated by infrared radiation, attenuationsof the illuminating beam on the order of 90% or greater would beexpected within distances as small as the dimension of the cells.Furthermore, such attenuation also would affect the radiation scatteredby the cells by severely distorting and attenuating the scattered wavesemerging from the suspending liquid.

It is not surprising, therefore, that those skilled in the art ofdifferential light scattering measurements and infrared radiationapparently long ago dismissed the possibility of making meaningful DLSmeasurements from such large organic particles at these wavelengths.Attenuation by a water sheath or even the water within airborne cellsthemselves appears superficially to all so skilled to preclude thepractical application in infrared radiation. Recognizing that thescattering of radiation by objects in the resonance region is notgenerally governed by the geometrical optics attenuation relations, theexpected scattering properties of heavily water-laden particles in theinfrared resonance region have been determined and are described laterin this disclosure. They demonstrate conclusively that such particlesmay be distinguished by their DLS patterns provided that they aresurrounded by a gas such as air that does not strongly absorb infrared.Because of this, it is preferred to perform the scattering measurementof a cell in a gaseous environment. While air is employed as the gas inthe preferred structure now to be described, it should be understoodthat any of a variety of other gases could be employed if desired; orthe analysis even could proceed in a vacuum provided the particles ofinterest maintained their structure in such an environment; or if aninfrared transparant fluid were found, the measurements could even beperformed therein; or if only a relatively thin layer of water coatedthe cells, the measurements could be performed with no problems.

SUMMARY OF THE PREFERRED EMBODIMENT

As noted, the practical application of the resonance scatteringphenomena to large organic particles such as mammalian cells or pollenparticles requires the use of polarized infrared radiation at awavelength of about 10 μm. In summary, the cells or particles are firstaerosolized, then transported through a detector array by means of alaminar flow of dry gas. The detector array, consisting of N≃2ρ_(max) +4discrete detector elements, preferably subtends an angle of about 100°of the DLS pattern produced by the individual particle as it passesthrough a collimated laser beam, the beam preferably being coplanar andat right angles to the array elements. Certain detectors, may have to becryogenically cooled and insulated from the dry gas stream by means ofan insulating cylinder including windows made of an infrared transparentsubstance such as germanium.

As a particle passes through the laser beam it produces a pulsedspherical wave of duration D/V, where D is the beam diameter and V isthe particle/stream velocity. The individual detector elements convertthe pulse they receive into an electrical signal that is amplified andstored, preferably in digital form after conversion, in a computermemory or on a tape. These N≃2ρ_(max) +4 stored signals may be used toreconstruct the continuous DLS pattern from the particle byinterpolation procedures using Tchebyshev polynomials, or relatedprocedures, or the stored signals may be used directly. The angularspacing of the individual elements may be equidistant, though forcertain types of subsequent analyses it may be preferable to space themaccording to the locations of the N roots of the Tchebyshev polynomial,T_(N) (X), as will be apparent from this disclosure to those skilled insuch analyses, where N is the number of detector elements, X=[θ-(θ₂+θ₁)/2]/[(θ₂ +θ₁)/2], and θ₁ <θ₂ define the angular range over which theDLS pattern is to be recorded.

Once the DLS patterns, or sets of array data points from which suchpatterns may be reconstructed, have been stored, these patterns may beanalyzed by means of various algorithms so that each particle may beidentified or otherwise suitably characterized. Most practicalalgorithms have been found to be based on a characterization procedurethat first groups particles into sets of equal size. The average size ofthe particles may be sufficiently estimated by counting the number ofDLS peaks between two angular limits or by determining the angularposition of a specific peak with respect to the forward direction. Sincesuch a deduced size parameter is not a conclusive means foridentification or differentiation, its exact value is unimportant solong as particles of the same effective size are all compared with oneanother. Having established that all particles in a given set areeffectively of the same size, e.g. have the same number of peaks betweentwo angular limits, the particles may be structurally and physicallydistinguished from one another by various sorting algorithms. Thesealgorithms may characterize DLS patterns by comparing various ratios ofa given DLS pattern. For example, the ratios of the heights of the DLSpeaks in a given angular range to the height of the first peak in thatrange are a useful set of differentiation parameters, as will bedetailed subsequently. Other ratios would include various peak-to-valleyvalues as well as ratios involving functionally more complex terms basedon the various peak heights and valley depths present. All such ratiosare functions of the scattering particle's dielectric structure and maybe used therefore to characterize each particle. Once such ratiocharacterizations have been achieved for each of the size groups ofparticles present, these may be further analyzed by means of a storedcatalogue of such ratios contained in the computer memory of thesystem's analytic processor. The distribution of such scattering ratiosas a function of particle size set represents another important meansfor identifying, characterizing and differentiating particles.

DESCRIPTION OF A PREFERRED APPARATUS

As shown in FIG. 1, a schematic illustration of the various means orelements of a preferred apparatus, a suspension of cells to be analyzedis supplied to a cell sorter 2 similar in construction to that describedby W. A. Bonner, et al. in "Fluorescence activated cell sorting"appearing in Rev. Sci. Instruments, 43, pg. 404 et. seq. (1972). Thiscell sorter, shown in more detail in FIG. 2, separates the liquidsuspension of cells into a series of discrete droplets, the separatedroplets being of a size small enough to contain no more than onemammalian cell or particle. As set forth in the noted description, thesedroplets are electrostatically charged, illuminated by a light beam todetermine if they contain a particle, then sorted by electrostaticdeflection to produce a droplet stream containing only droplets thatincorporate mammalian cells. This droplet stream 4 is supplied to adetector system 6, also illustrated in FIG. 2, that illuminates eachdroplet with a polarized monochromatic beam, preferably an infraredlaser of 10.6 micrometers wavelength. The illumination scattered by eachcell in succession is measured by a sensor system, preferably an arrayof sensors, incorporated in the detector 6. The output electricalsignals of each detector then may be transmitted to and stored in arecorder 8 such as a magnetic tape recorder. The stored data then may beanalyzed by a computer analyzer. After sufficient particles have beenanalyzed, the computer may summarize the results and provide an outputin tape, disc, or hard copy form, as will be described subsequently.

FIG. 2 illustrates in more detail the sorter and detector portions ofthe preferred apparatus. As described in the article by W. A. Bonner, etal., the liquid suspension of cells passes from an orifice 22 in tube 24producing a series of droplets 26, each droplet being charged by anionization source. These discrete droplets are illuminated by a beam 28,the scattered intensity from each droplet being sensed by a detector 30,amplified, and transmitted to an analyzer 32 which controls and suppliesan electric potential to a set of electrostatic deflection plates 34.The stream of droplets passes between the plates of this electrostaticdeflection system. As described in the article by Bonner, et al., thescattering produced by each successive droplet and measured by thedetector 30 is analyzed to determine the presence or absence of a cellin that droplet, the analyzer energizing the set of deflection plates toelectrostatically deflect from the stream those droplets 36 which do notcontain a cell. The remaining droplets 38 which do contain a cell orparticle pass to the detector system.

The detector system incorporates in a housing or scattering chamber 42an array of sensors 44, an inlet 46, and an exhaust opening 48. Aradiation source 50, preferably a laser, produces a beam ofmonochromatic, preferably plane polarized, infrared radiation 52. Thisbeam passes into the housing 42 through an opening 54 and from thehousing through an opening 56, thereafter passing into a Rayleigh hornor light trap 58. Preferably, the axis along which the beam passes liesin the plane of detector sensor array 44, and is orthogonal to the axisalong which the stream of droplets pass, although this relationship isnot essential to the operation of the apparatus as is notedsubsequently. The detector array preferably is a liquid nitrogen cooledmulti-element mercury-cadmium-telluride [Hg Cd (Te)] array such asproduced by Honeywell Corporation Radiation Center, although othersuitable arrays are produced by other groups such as Arthur D. Little &Co. and the Santa Barbara Research Center, a subsidiary of the HughesAircraft Corporation. Such detectors have very high detectivities makingthem most suitable for this measurement. However, the requirement tocool them cyrogenically may be inconvenient or undesirable in someapplications. Accordingly, pyroelectric detectors which may be operatedat normal room temperatures are also most suitable for measurements inthe vicinity of 10 μm. Although the detectivities of such detectors areless than those of the cooled Hg-Cd(Te) type by a factor of about 100,the availability of almost unlimited power radiation sources, such asCO₂ lasers insures a more than adequate scattered signal. Pyroelectricdetectors are also considerably less expensive than their Hg-Cd(Te)counterparts, thereby promising greatly reduced fabrication andoperating costs. A collection of papers by Honeywell Corporation staffis particularly appropriate. This "Compendium of Honeywell Publicationson Pyroelectric Detectors and Materials" is available from the HoneywellCorporation Radiation Center in Lexington, MA. It includes many relatedpapers, both published and unpublished, pertinent to the preferredsensor array, papers such as:

S. T. Liu, J. D. Heaps and O. N. Tufte, "The pyroelectric properties ofthe lanthanum-doped ferroelectric PLZT ceramics," Ferroelectrics 3, pgs.281 through 285 (1972), and

A. van der Ziel and S. T. Liu, "Noise sources in pyroelectric radiationdetectors," Physica 61, pgs. 589 through 593 (1972).

Preferably, the individual elements of the array are spaced from oneanother about 2 mm, 10 to 50 discrete elements being distributed over anarc of approximately 100° extending from a scattering angle of 30° to ascattering angle of 130°. If the elements are Hg-Cd(Te) detectors, suchan array must be cryogenically cooled. To this end, a source of liquidnitrogen at cryogenic temperature [about 77° K. for a Hg Cd (Te)detector array] is supplied to a jacket 62 (FIG. 3) incorporated inhousing 42 and surrounding the detector array 44. Preferably, the innersurface of the cylinder about which the array is spaced has a radius ofabout 1 centimeter. If the detectors are to be cryogenically cooled,they must be isolated from the air environment by means of a vacuumbetween the detectors and the flowing air stream. This is most readilyachieved by means of a concentric inner structure, 64, the volumebetween the array 44 and the innermost well 45 being evacuated. Thisinner structure includes suitable windows made of germanium or any otherinfrared transparent substance.

The liquid volume of the droplets about the cells is evaporating as thecells pass from the cell sorter in and through the detector. Preferablythe humidity of the transporting air stream is adjusted so that theliquid enveloping the cell in the droplet is just evaporated duringtransit of the cell from the sorter to the detector providing a freeairborne cell for illumination in the detector region. Thus, theatmosphere flowing along with the cells will tend to be rather humid.Should this humid atmosphere encounter cryogenic temperatures, or evenbe cooled to any significant degree, condensation may occur. Suchcondensation would significantly affect the light scatteringmeasurement. To avoid such problems, it is preferred both to minimizethe volume of the atmosphere flowing into the detector with the streamof cells and also to surround this atmosphere and stream of cells withan insulating sheath of dry gas. Also, as noted, the aerosolizationprocess should be separated sufficiently from the detector to permit allaccreted water to evaporate yet not dehydrate the cells. Ideally, theliquid surrounding each cell shall have evaporated just before the cellenters the detector array, yet no liquid internal to the cell have hadan opportunity to transpire through the cell membrane. The sheath of dryair is provided from a source of dry air 69 through a collar 66 whichintroduces it into the detector as a laminar flowing column about thestream of cells, this column of dry air isolating the stream of cellsfrom the infrared window 64. The column of dry gas and the stream ofcells it surrounds is exhausted through opening 48 in the housing,passing through a conduit 68 at negative pressure to a receptacle (notshown).

As best shown in FIG. 3, when each individual cell passes through thebeam of radiation 52 it scatters that radiation, some of the scatteringbeing intercepted by each discrete detector in the detector or sensorarray 44. The resultant discrete electrical signals produced by eachdetector in the array are transmitted through the cable 8 to asubsequent electronic analysis and recording system.

Especially when considering mammalian cells, should the data includesignificant structural and surface differences in addition to size andrefractive index variations, it will present an analyticalinterpretation task well beyond the means of current technology. Sucheasily can be the case when large particles are illuminated with visiblelight. Most importantly, the plethora of detail in such differentiallight scattering patterns derived using visible light appears to bear nosimple, interpretable relation to the amount of physical data that maybe deduced from them. Extracting the salient particle characteristicfrom such patterns, if possible, presents at best an immense task.

It might be argued, however, that one could record the differentiallight scattering patterns with much less angular resolution and therebyminimize the abundance of data relative to the physical parametersinvolved. However, as is revealed by an examination of such patterns aspresented, for example, in the Applied Optics paper by Blau, et al.,even the envelopes of such differential light scattering patterns changesignificantly for miniscule changes in the scattering particle'sstructure. Thus, decreasing the angular resolution will not in itselfresult in a satisfactory improvement of the parameter deduction problem.

Illustrated in FIG. 4 are computer-generated differential lightscattering patterns for cells with a high water content illuminated by avertically polarized (electric vector in a plane orthogonal to the planeviewed by the sensors) beam of monochromatic radiation having awavelength of 10.6 micrometers. The cells in FIG. 4a have a radius of 10micrometers, and the cells in FIG. 4b have a radius of 20 micrometers.The former size would be similar to leucocytes, whereas the latter wouldbegin to approach that of squamous cells. In these examples, fourdifferent compositions have been chosen, compositions that wouldcorrespond to slightly different amounts of protein in the particles.Since the presence of additional protein will increase only the realpart of the refractive index of the particle, the four examples selectedexhibit only slight changes in this real part of their averagerefractive indices; to wit:

A: n=1.176+i0.084 (This is the approximate value of the refractive indexof pure water at 10.6 μm; i.e. an `empty` droplet)

B: n=1.20+i0.084

C: n=1.25+i0.084

D: n=1.30+i0.084

The respective curves in each figure are in the order indicated by therespective letters. FIGS. 4c and d illustrate patterns corresponding toparticles identical to those yielding FIGS. 4a and b, respectively,except that the patterns of FIGS. 4c and d result from horizontalpolarization (electric vector in a plane coincident with the planeviewed by the detectors) of the illuminating infrared radiation.

As this data illustrates, relatively simple structural changes in thescatterer produce relatively simple changes in the differential lightscattering pattern. For the smaller particles, the horizontallypolarized scattered intensity at around 40° (FIG. 4c) relative to theintensity at 50° provides a simple yet excellent measure of the physicaldifferences of the postulated cells. Similar simple and obviousdifferences for the larger cells are shown in FIG. 4d. Accordingly,while differential light scattering patterns produced by such cells whenilluminated with shorter wavelengths of visible radiation are extremelycomplex and yield a plethora of data far in excess of the number ofphysical parameters involved, the corresponding patterns obtained atlonger infrared wavelengths in which the particles and illuminatingradiation are in the resonance region result in data that is far easierto understand and to interpret. This permits particles to be identified,differentiated, characterized and analyzed accurately with relativelysimple, readily available computerized equipment.

It should be noted particularly that the particles producing thepatterns illustrated in FIGS. 4a-d have refractive indices close to thatof water in the infrared, i.e., have a high water content, and aretherefore highly absorbing in the infared. Nevertheless, theirscattering patterns show distinctive differences for relatively smallthough significant composition changes. Because of the large imaginarypart of the refractive index, such differences would not generally beexpected. Therefore, those traditionally skilled-in-the-art would avoidusing infrared radiation and not even attempt to check their intuitivelywrong expectations by means of analyses such as these.

A preferred analytical system is shown in FIG. 5. As previouslyexplained, when each cell of the cell suspension passes through the cellsorter, it results in a differential light scattering pattern beingimpressed upon and sensed by the detector array 44. In one embodiment ofthe system, shown in FIG. 1, these successive differential lightscattering patterns, or more precisely the successive intensitymeasurements produced by the array of detectors, may be recorded by arecorder 8, such as a magnetic tape recorder, the recorder providing achannel for each detector of the array. Subsequently, the intensityvariations produced as the recorded output may be analyzed first todetermine the peak intensity of each detector for each cell to beanalyzed, the peak intensities being combined to produce differentiallight scattering patterns such as shown in FIG. 4.

These patterns, or portions of these patterns, may be analyzed in any ofvarious ways to identify, classify, and characterize the cells whichproduce them. For example, referring to FIG. 4b, the particles ofidentical size but of slightly different refractive indicescorresponding to different protein compositions which produced thepatterns presented in that figure may be distinguished from one anotheron the basis of the secondary peak amplitudes relative to the amplitudeof the first peak, the first peak being that at approximately 25°. Thetable below lists these ratios for the first four peaks.

                  TABLE I                                                         ______________________________________                                        DLS PEAK RATIO FOR FIG. 4b                                                    Peak PARTICLE    A        B      C      D                                     ______________________________________                                        1                1        1      1      1                                     2                .33      .30    .21    .19                                   3                .13      .089   .078   .067                                  4                .056     .033   .026   .022                                  ______________________________________                                    

As this table readily illustrates, based upon such different peakratios, particles of the same size but of slightly different physicalproperties easily may be differentiated. Thus, to discriminate amongparticles of different size and different structure, such as are presentin a suspension of the mammalian cells, the differential lightscattering patterns which result from the various individual particlesfirst may be separated by the number of peaks they present, thisseparation collecting into groups those light scattering patternsarising from particles of approximately the same size. After such arough size grouping, those particles of approximately the same size maybe compared with one another. As a first differentiation of theseparticles, the intensity of the first peak may be used as a standardvalue and the ratio of the second peak to this first peak intensitymeasured and employed as a more accurate differentiation than simply asize differentiation. Indeed, as has been noted, leucocytes include anumber of different cell types, cell types which range in size fromlymphocytes at approximately eight micrometers in size to granulocytesat approximately 18 micrometers. The size of appreciable numbers ofthese cells will be approximately the same, say in the order of 12 to 13micrometers. Such particles may have approximately four to five peaks intheir light scattering pattern when illuminated with verticallypolarized monochromatic infrared radiation. If the ratio of the first tothe second peak is employed to differentiate these particles in asimple, two-dimensional analysis, various overlapping Gaussiandistributions will result generally in accordance with the overlappingdistribution of leucocyte types in this smaller size range. To furtherdifferentiate these distributions, each successive peak ratio may beemployed in a multi-dimensional vector analysis. While such an operationmay be performed by hand, it is more convenient to employ a standardpattern recognition technique such as a typical multidimensional vectorspace partition analysis to group samples of similar characteristicsusing, for example, an appropriately programmed electronic computer.Such an analytical approach is well within the ability of one skilled inthe art and indeed today is performed routinely to classify complex dataemploying a multi-dimensional array.

When a large number of cells are to be analyzed, or for other reasons itis inconvenient to perform the cell identifcation and classification byhand as just described, an electronic system as shown in FIG. 5 may beemployed. In this system, the output of each detector of the detectorarray 6 is supplied preferably to a logarithmic amplifier 72.

By converting each detector output signal which is a linear function oflight intensity, produced at each detector, into a logarithmic value asachieved by a logarithmic amplifier 72, the dynamic range of the systemis broadened considerably without increasing the digital data handlingrequirements. In addition, manipulating and comparing the data issimplified appreciably, since, for example, to determine ratios it isonly necessary to subtract logarithms. On the other hand, with the rapidadvent of inexpensive digital calculators, the alternative use of linearamplifiers supplemented by more complex arithemetic operations would beequally attractive. The response of these separate logarithmic or linearamplifiers may be standardized initially by causing light of a uniformintensity to strike all the detectors of the array simultaneously andthen adjusting the amplifiers so that all produce the same output.

Detector standardization may not be required if the absolute differencesbetween the gains of the individual detectors are measured and storedfor subsequent arithmetic correction. Alternatively, any intensity setproduced by a single particle could be used as a reference set by whichall subsequent sets could be normalized or corrected.

Since the linearity of each detector of the infrared sensitive arraydiscussed earlier is excellent, the outputs of the logrithmicamplifiers, by means of the standardization adjustment, will accuratelyrepresent the logarithm of the respective intensities of theillumination striking the respective detectors. These logarithmicamplifiers may be of the type made by Analog Devices, Inc., device no755. These respective outputs are transmitted to sample and peakdetectors 74 such as manufactured by Burr Brown, device no. 4084.

A discriminator 76 is connected to the logarithmic amplifier supplyingthe output of the lowest angle detector. As the intensity produced bythis low-angle detector varies in response to passage of the particle,the variation is noted by the discriminator. The discriminator 76 alsois connected to the peak detectors 74 and holes them in a clear stateuntil the previous analytical cycle is completed and the next cyclebegins. This is triggered by the intensity of the output of thelogarithmic amplifier connected to the lowest angle detector exceeding apredetermined level sufficient to indicate that a particle is passingthrough the beam of monocromatic radiation. As the particle passesthrough this beam, the output of each detector varies, reaching amaximum value which is stored by the peak detector 72 connected to it.These stored intensities correspond to the intensities of thedifferential light scattering pattern produced by that particle at thevarious successive angles of the detectors. As the source particlepasses out of the laser beam, the intensity sensed by the lowest angledetector diminishes. The discriminator 76 responds to this decreasingmagnitude and actuates a control logic system 78 by means of aconnection 80. The control logic system 78 in turn actuates an analog todigital conversion device 82 which is sequentially connected by means ofa multiplexer 34 to each peak detector 74. Such a multiplexer andconversion device may be, for example, that offered by Burr Brown asdata acquisition unit MP 8126.

As a result of this processing, the logarithmic analog signal stored ineach of the peak detectors is sensed and converted to a digitalrepresentation. This representation is transmitted to a memory system86, preferably formed by emitter coupled logic components such asmanufactured by Motorola, where it is stored in sequence with the othersuccessive digital representations. Accordingly, stored in the memoryunit is a digital representation of the peak value of the scatteredlight intensity sensed by each successive detector in array 44. Afterthis operation, the control logic system 78 signals the discriminator 76to permit new data to be accepted.

In the preferred embodiment, the memory unit 86 is connected to amicroprocessor 88. The microprocessor examines the data by cyclingthrough the digital information stored in the memory to determine thenumber of peaks present, employing mathematical interpolation if thenumber and spacing of the detectors are insufficient to provide thedesired accuracy, this examination resulting in a digital sequenceoutput representing the number, location, and values of such peaks. Morespecifically, the microprocessor analyzes the data to determine, forexample, the ratio of the intensity of the second peak measured to theratio of the intensity of the first peak measured, producing a firstratio, the digital representation of which is held by themicroprocessor. In similar fashion, the microprocessor processes thedata stored in the memory unit to determine the successive peak ratios,thereby resulting in a digital output that indicates, first, the numberof peaks in the differential light scattering pattern produced by theparticle just sensed by the detector array, then the peak ratios of thisparticle such as those ratios set forth in Table I.

The microprocessor 88 and the control logic system 78 both may utilizebipolar high-speed bitslice microprocessors such as those manufacturedby Motorola or Texas Instruments, for example Motorola microprocessorno. MC10800. This microprocessor is controlled by a programmableread-only memory to perform the sequential analysis as just described orany other desired analysis.

The resulting stream of digital data may be recorded such as on a discdata storage unit 90, or it may be displayed on a video terminal 92, orcomplied as a hard copy output by printer 94, or it may be stored in alarger memory. While the storate unit, video terminal, and printer maybe connected directly to the microprocessor, preferably further analysisof the data is performed by a minicomputer 96, the central processor ofwhich first causes the data to be transmitted to the disc data storageunit 90. Then it analyzes the stored data by, for example, amultidimensional vector space partition analysis program or othersuitable sorting algorithm as previously noted to construct a videodisplay on terminal 92 of the various cell types present in thesuspension supplied to the system, this display being printed inresponse to a user command by printer 94. The minicomputer 96 is aDigital Equipment Corporation PDP 11-20 unit, although various othercomputer systems will quite satisfactorily perform this analysis as iswell known to those skilled in this art.

Many previous systems employing a detector or a detector array tomeasure the light scattered by an object over a substantial arcemphasize the importance of maintaining the detector or detector arrayat a constant radial distance from the object throughout the measurementarc. It is preferred to employ a detector array in the apparatus of thisinvention, as previously noted. This requirement of a constant radialdistance imposes significant limitations upon the array. Not only mustit be fabricated to form an arc of the appropriate radius and length,but also in accordance with prior teachings, the sensitivity of eachelement of the detector array should be quite uniform. Such limitationssignificantly increase the cost of the array and the cost of theassociated electronics system required to achieve and maintain suchuniformity.

The reason for this requirement is that light intensity diminishesinversely as the square of its distance from the scattering object.Thus, if a detector array is used, and all of the detectors in the arrayare not all exactly the same distance from a uniformly radiating object,unequal intensities will fall upon the elements of the array. Further,the surface area of the elements should be exactly equal so that theyintercept the same solid angle of radiation, all to achieve a uniformityof response of each detector in the array to uniform scattering by theilluminated object. Only by realizing such uniformity will lightscattering patterns such as illustrated in FIGS. 4 be achieved.

An important aspect of the present invention is the teaching that suchuniformity need not be present in the detector array. Indeed, thedetector array may consist of a number of linear segments deposed aboutthe interior of the housing, the linear segments being configured asshown in FIGS. 6. Of course, a greater or lesser number of segments maybe employed if desired, and they may be configured in various othermanners. Each adjacent element or detector of the array, being at adifferent radial distance from the scattering object, will interceptlight scattered in a different solid angle. In addition, these detectorsneed not be in the same plane. These differences and others in the arrayall will contribute to a significant distortion of the intensity oflight sensed by the detectors constituting the array. This distortioncan be considered to be a simple transformation of the undistortedscattering pattern. However, such a transformation need not result inerroneous characterizations of the analyzed cells. The light scatteredby each substantially identical cell will result in a substantiallyidentical, though transformed, differential scattering pattern beingsupplied to the processing system. Similarly, cells of differentcharacteristics will result in correspondingly different scatteringpatterns similarly transformed and supplied to the processing system.For discrimination, characterization, or identification purposes, it isonly necessary to achieve a consistency among the array elements andtheir responses transmitted to the processing system from identicalparticles illuminated in their transit through the detector housing, anda difference between the transformed scattering patterns applied to thepreprocessor for substantially different cells being illuminated in thedetector housing. Even though a detector array composed of variouslinear detector segments, as shown in FIGS. 6, results in atransformation of the true scattering pattern, the transformed patternstill results in substantially identical light scattering patterns beingsupplied to the preprocessor as the result of substantially identicalcells being illuminated, and substantially different patterns beingapplied to the processing system for substantially different cells.Thus, the system is still capable of correlating substantially identicalcells and distinguishing among non-identical cells. For this reason,significant savings in cost and simplification in structure of thedetector array is realized in the disclosed apparatus while stillattaining a major objective of the invention: rapid, unambiguousdifferentiation, characterization and identification of mammalian cellsand other large particles such as pollens and fungal spores.

It should also be noted that the transformed DLS patterns that aremeasured for subsequent identification and discrimination of the sourceparticles may be of many different types and measured in many differentways. Though desirable, even an array is not essential for this purposeas there are other alternatives for measuring and recording DLSpatterns. If the source of illumination is co-linear with the particlestream, a single detector will synthesize the DLS pattern of acontinuous array. Such an arrangement is illustrated in FIG. 7. A beamof illumination 102 is reflected from a mirror 104 shaped to direct thebeam along the path of fall of the cells 106. As each cell passes intothe detector housing 108, it comes into the field of view of the singledetector or sensor 110. During the transit of the cell through thehousing, its scattered illumination is viewed continuously by detector110. Thus, the output of the detector will be a continuousrepresentation of the illumination scattered by the cell from the lowestangle viewable by the detector as the cell passes into the housing tothe highest angle viewable as the cell exits from the housing. Thisrepresentation, when plotted as a function of time (and thus ofscattering angle) will be the differential light scattering pattern ofthe cell and may be employed in the analytical system previouslydescribed. As the cell approaches mirror 104 it is deflected by an airjet 112 into a reservoir 114.

Another way to measure differential light scattering patterns employs asingle, rotating detector. If a single detector may in effect be made torotate about the particle in a period shorter than the particle'stransit time through the perpendicular illuminating beam, a sufficientDLS pattern may be obtained. Such a configuration is described in anarticle by Marshall, Parmenter and Seaver, Science, Vol. 190, October,1975, pgs. 375-377, "Precision Measurement of Particulates by LightScattering at Optical Resonance", particularly with reference to FIG. 3.Alternatively, the particles may be electromagnetically captured andscanned individually as shown by Phillips, et al. in their U.S. Pat. No.3,754,830.

For subsequent mathematical analysis, any such DLS pattern, or sectionthereof, may be converted to a digital representation. As discussedearlier and reemphasized here, any such pattern may be sufficientlycharacterized by N coefficients where N is approximately≃ 2ρ, oralternatively by means of N discrete intensity values spanning theangular range of interest. For digital purposes it is probably mosteconomical to store such DLS patterns in terms of the N coefficients, bywhich means they may be reconstructed later, than the much greater setof numbers corresponding to the digital storage of DLS patterns obtainedfrom the synthetic continuous array derived from a single detectorconfiguration of the types described above.

While a preferred system and components have been disclosed, dependingupon the number of cells desired to be stored by the system per minute,slower and less expensive components may be employed, or fastercomponents may be required. Of course, the cycle time of thesecomponents also is related to the number of detectors in the detectorarray. For the system disclosed, using a detector array of 10 to 50sensors, a cell throughput rate may be achieved on the order of 1,000 to60,000 cells per minute, a rate more than adequate to equal or exceedmost cell sorting requirements. In addition to using faster components,higher sorting rates also may be achieved by using multiple memories andmicro-processing systems, since in the stream of cells the average cellrate will be appreciably less than the maximum cell rate due to the factthat a number of droplets will contain no cells and will be deflectedfrom the cell stream by the cell sorter.

While preferred embodiments of the invention have been disclosed anddescribed, as previously noted, various other embodiments may bepreferred by others skilled in this art. Accordingly, the scope of theinvention is not limited to the preferred embodiment.

What is claimed:
 1. A process for analyzing the cells of a sizesubstantially larger than a wavelength of visible light, the cells beingin a liquid suspension, the process comprising the steps of:Aerosolizingthe suspension of cells to produce a series of droplets, some of whichcontain a cell; Separating those droplets containing a cell from theother droplets which do not contain cells; Illuminating in sequence theseparated cells with a beam of monochromatic radiation of a wavelengthwhich, when compared to the size of the cell, is in the resonanceregion; Detecting radiation intensities of a given polarizationscattered by each illuminated cell to obtain a differential scatteringpattern characterizing the size, shape, and dielectric properties ofeach cell; and Recording the detected patterns to characterize theilluminated cells.
 2. A process for analyzing cells as set forth inclaim 1 in which the detected intensities are analyzed by:Determiningthe extrema within the set of intensities detected for each pattern;Counting the number of extrema within the set of intensities detected toproduce a total count for each pattern; Associating such total countwith an average size interval; and Comparing each detected pattern witha set of reference patterns of the associated average size interval tocharacterize or associate each particle with a known reference patternby this comparison.
 3. A process for analyzing cells as set forth inclaim 1 in which the radiation employed to illuminate the cells is of awavelength that is no greater than substantially equal to the size ofthe cells and no less than approximately one twentieth the size of thecells.
 4. A process for analyzing cells as set forth in claim 3 in whichthe detected patterns are analyzed byLocating the angular locations ofthe extrema of the pattern represented by the angular sequence ofdetected signals for each cell to obtain sets of extrema data, one setfor each cell; Grouping the sets of extrema data into particle sizegroups based on the number of extrema in a selected angular range;Normalizing each extrema intensity value relative to the intensity valueof a reference extrema value; and Comparing such normalized values witha reference set of values to associate each analyzed cell with one of areference set of cells.
 5. A process for analyzing cells as set forth inclaim 4 in which the reference set of values are obtained by analyzing asuspension of known cells by a process comprising the stepsof:Aerosolizing the suspension of cells to produce a series of droplets,some of which contain a cell; Separating those droplets containing acell from the other droplets which do not contain cells; Illuminating insequence the separated cells with a beam of monochromatic radiation of awavelength which, when compared to the size of the cell, is in theresonance region; Detecting radiation intensities of a givenpolarization scattered by each illuminated cell to obtain a differentialscattering pattern characterizing the size, shape, and dielectricproperties of the cell; and Recording the detected patterns tocharacterize the illuminated cells.
 6. A process for analyzing cells asset forth in claim 5 including the step of:Evaporating the liquidsurrounding each cell droplet prior to illuminating in sequence theseparated cells.
 7. A process for analyzing cells as set forth in claim6 in which the cells are organic cells from the group consisting ofmammalian cells, fungal spores, and pollen.
 8. A process for analyzingcells as set forth in claim 5 in which the radiation intensities aredetected by elements of a detector array at a sufficient number ofangular locations relative to the direction of the illuminating beam toderive a pattern representative of a differential scattering pattern. 9.A process for analyzing cells as set forth in claim 8 where the numberof locations N≃[2πD_(max) n_(o) /λ_(o) +4]θ/˜° where:θ is the angularrange in degrees spanned by the detectors; n_(o) is the refractive indexof the medium in which the measurement is made; D_(max) is the largestcell or particle diameter of the ensemble being examined; and λ_(o) isthe vacuum wavelength of the incident radiation.
 10. A process foranalyzing cells as set forth in claim 9 in which the angular locationsof the N detectors of the detector array are given by the N roots of theTchebychev polynomial T_(N) (X) where: ##EQU1## and θ₁ <θ₂ are the twolimiting angles defining the angular range of the pattern to bemeasured.
 11. A process for analyzing cells as set forth in claim 5 inwhich the reference set of values are obtained by examining each dropletin the suspension of known cells to detect the presence of a cell in itprior to the separating step.
 12. A process for analyzing cells as setforth in claim 1 in which each droplet is examined to detect thepresence of a cell in it prior to the separating step.
 13. An apparatusfor analyzing cells of a size substantially larger than a wavelength ofvisible light, the cells being in a liquid suspension including:Meansfor aerosolizing the suspension of cells to produce a stream ofdroplets, some of which include a cell; Means to separate those dropletsin the suspension which include cells from the other droplets which donot include cells; Means individually illuminating the separate cellswith a beam of monochromatic radiation, the radiation being of awavelength which, when compared to the size of the illuminated cell,produces resonant scattering; Means detecting the intensities of a givenpolarization scattered by the illuminated cell to obtain a differentialscattering pattern characterizing the size, shape, and dielectricproperties of the cell; and Recording the detected pattern for eachilluminated cell to thereby characterize each cell.
 14. An apparatus asset forth in claim 13 in which the radiation intensities are detected byelements of a detector array at a sufficient number of angular locationsrelative to the direction of the illuminating beam to derive a patternrepresentative of a differential scattering pattern.
 15. An apparatus asset forth in claim 14 in which the number of locations N≃[2πD_(max)n_(o) /λ₀ +∝]θ/1/2° where:θ is the angular range in degrees spanned bythe detectors; D_(max) is the largest cell or particle diameter of theensemble being examined; and n_(o) is the refractive index of the mediumin which the measurement is made. λ_(o) is the vacuum wavelength of theincident radiation.
 16. An apparatus as set forth in claim 15 in whichthe spacing of the N detectors of the detector array are given by the Nroots of the Tchebychev polynomial T_(N) (X) where: ##EQU2## and θ₁ <θ₂are the two limiting angles defining the angular range of the pattern tobe measured.
 17. An apparatus as set forth in claim 14 including:Adetector housing; Means detecting the scattered intensities comprisingan array of detectors deposed about the interior of the housing; andMeans directing aerosolized cells individually through the detectorhousing, the illuminating beam passing through the detector housing andilluminating the individual cells during their transit through thehousing.
 18. An apparatus of claim 17 in which the detectors are notequally spaced in angle.
 19. An apparatus of claim 18 in which thespacing of the N detectors of the detector array are given by the Nroots of the Tchebychev polynomial T_(N) (X) where ##EQU3## and θ₁ <θ₂are the two limiting angles defining the angular range of the pattern tobe measured.
 20. An apparatus as set forth in claim 17 in which theindividual detectors of the detector array are at different radialdistances from the point at which a cell in the housing is illuminatedby the beam.
 21. An apparatus as set forth in claim 20 in which thedetectors in the array view radiation along axes deposed in a singleplane, this plane including the axis of the illuminating beam.
 22. Anapparatus as set forth in claim 21 in which the linear detector arrayconsists of at least one set of planar detector elements all beingdeposed in substantially the same plane.
 23. An apparatus as set forthin claim 13 in which the wavelength of the illuminating beam is nogreater than substantially equal to the size of the illuminated cellsand no less than substantially one twentieth the size of the illuminatedcells.
 24. An apparatus as set forth in claim 13 including means foranalyzing the set of intensities detected comprising:Means determiningthe extrema within the set of intensities detected for each pattern;Means counting the number of extrema within the set of intensitiesdetected to produce a total count for each pattern; Means associatingsuch total count with an average size interval; and Means comparing eachdetected pattern with a set of reference patterns of the associatedaverage size interval to characterize or associate each particle with aknown reference pattern by this comparison.
 25. An apparatus as setforth in claim 24 including:Means comparing each of the extrema valuesin the extrema set to an associated reference extrema value to yield aset of normalized data; and Means comparing the detected and referencepatterns comparing normalized data.
 26. An apparatus as set forth inclaim 25 including:A detector housing; Means detecting the scatteredintensities comprising an array of detectors deposed about the interiorof the housing; and Means directing aerosolized cells individuallythrough the detector housing, the illuminating beam passing through thedetector housing and illuminating the individual cells during theirtransit through the housing.
 27. An apparatus as set forth in claim 26in which the detectors in the array view radiation along axes deposed ina single plane, this plane including the axis of the illuminating beam.28. An apparatus as set forth in claim 27 in which the detectors of thearray are generally planar in shape, the detectors being located atoffset irregular positions about the illuminated cell and atsubstantially different radial distances from said cell, at least someof the detectors being coplanar with the incident radiation beam.
 29. Anapparatus as set forth in claim 13 including means to examine eachdroplet in the aerosolized suspension to detect the presence of a cellin it prior to separation of the droplets.
 30. An improved detectorsystem for a photometer, the photometer including an illuminating beam,means for locating the sample in the illuminating beam to produceradiation scattered by the sample, the improved detector systemincluding means locating the detector generally along a linear path tomeasure the scattered intensity at offset angular locations about saidsample, said means locating the detector to measure the scatteredintensity at substantially different radial distances from said samplelocation, the detector locations at lower acute scattering angles beingat substantially greater radial distances from said sample location thanthe detector locations at higher acute scattering angles.
 31. Animproved detector system as set forth in claim 30 in which the detectorcomprises an array of detector elements, different elements producingsubstantially different electrical responses to scattered radiation,individual detector elements being located to measure the scatteredintensity at offset angular locations about said sample location and atsubstantially different radial distances from said sample location. 32.An improved detector system array as set forth in claim 31 including aseries of discrete detectors, the detectors being aligned to view alongaxes all in a plane, which includes the axis of the illuminating beam.33. An improved detector system array as set forth in claim 32 in whichthe discrete detectors are generally planar in shape, at least some ofthe detectors being aligned in the same plane.